The Fas antigen (Fas) is a member of the tumor necrosis factor receptor (TNFR) superfamily that induces rapid apoptotic cell death upon binding of the Fas ligand. Fap-1 is a recently-identified protein tyrosine phosphatase that binds to the Fas negative regulatory domain and inhibits Fas-induced apoptosis; this inhibition requires the Fap-1 catalytic domain. Present data support the hypothesis that Fap-1 inhibits Fas-induced apoptosis by dephosphorylating tyrosine residues on specific proteins whose tyrosine phosphorylation is required for Fas-induced apoptosis. The objective of the proposed research is to elucidate the mechanism underlying Fap-1 inhibition of Fas-induced apoptosis by identifying Fap-1 PTP substrates. This will also illuminate the role of tyrosine phosphorylation in Fas signalling. Specifically, the goals are to (1) first ascertain the functional importance of the physical interaction of Fap-1 with Fas; (2) identify specific proteins that become tyrosine phosphorylated following Fas activation (by immunoblotting and phosphotryptic peptide mapping); (3) identify which of these tyrosine-phosphorylated proteins are then specific substrates of Fap-1 both in vivo and in vitro; and (4) map the tyrosine residues that are dephosphorylated to Fap-1 in the identified Fap-1 substrates. Loss of the regulation of cell death, resulting in the inability of a cell to undergo apoptosis when required (for example, if Fap-1 is constitutively activated) may contribute to unregulated cell growth and neoplasia. Furthermore, since many anti-tumor therapies (ie, chemotherapeutics) are cytotoxic through apoptotic pathways, functional analysis of Fap-1 protein in targeted tumor cells may aid in understanding and tailoring their apoptosis-sensitivity.